Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trx-transfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.     

The induction of DNA strand breaks in normal human skin fibroblasts exposed to solar ultraviolet radiation

The levels of apparent DNA single-strand breaks (ssb) were measured, following a 0-20 h incubation of normal human skin fibroblasts exposed to the solar uv wavelengths produced by a fluorescent sunlamp. The ssb were determined using the alkaline elution assay, which was performed either without proteinase K (proK) or in its presence, so as to eliminate any DNA-protein crosslinks that may be present in the cells. Cells were irradiated with either 3 kJ/m2 of sunlamp uv greater than 295 nm, 150 kJ/m2 of sunlamp uv greater than 315 nm, or 150 kJ/m2 of sunlamp uv greater than 320 nm. These treatments resulted in the production of 5-10 ssb/10(10) Da. For the two shorter wavelength irradiations, the levels of ssb decreased rapidly upon incubation of the cells. However, when the elutions were performed using proK, the number of ssb increased about twofold following a 2-4 h incubation. In contrast, the levels of ssb decreased in the sunlamp uv greater than 320 nm irradiated cells for elutions performed with or without proK. These results suggest that under certain irradiation conditions, ssb are formed in cells upon incubation, which are hidden by the crosslinking of protein to DNA.     

Modification of damaging effect of ultraviolet radiation on peritoneal macrophage membranes in mice

Different factors modificating damaging effect of middle-wave ultraviolet radiation (lambda max = 306 nm) on mice peritoneal macrophage plasma membranes were studied. It was shown that damaging effect of ultraviolet declined when the cells were simultaneously treated by red light (lambda max = 713 nm). This protective effect increased when a red component of light became greater and achieved almost 100% when it consisted 28.6%. It was also found that the decrease in intra- and extracellular pH led to the decrease in damaging effect of UVR. The increase of pH bring to an elevation of UVR destructive effect.     

Baicalin protects human skin fibroblasts from ultraviolet A radiation-induced oxidative damage and apoptosis

Reactive oxygen species (ROS) are an important factor in the development of skin photodamage after ultraviolet A (UVA) radiation. A flavonoid antioxidant, baicalin, can selectively neutralize super-oxide anion (O₂^?) while having no significant effect on ^?OH. Fibroblasts are a key component of skin dermis. In the present study, we investigated the protective effects of baicalin on human skin fibroblasts (HSFs) under UVA induced oxidative stress. Fluorescence microscopy and flow cytometry were used to assay the intracellular O₂^?, NO, ROS concentrations and the mitochondrial membrane potential. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular MDA, SOD, GSH, T-AOC, and 8-oxo-dG were also measured. Cellular apoptosis was measured by flow cytometry and caspase-3 detection. The results revealed that UVA radiation could cause oxidative stress and apoptosis in HSFs. Interestingly, the use of baicalin after UVA radiation signi?cantly reduced the level of intracellular O₂^?, NO, and ROS, stabilized the mitochondrial membrane potential, and attenuated production of MDA and 8-oxo-dG. These ef?ciently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage and apoptosis. In other words, baicalin decreased the excessive generation of intracellular ROS and NO, and elevated the cellular antioxidative defense, which eventually mitigate the UVA-induced apoptosis. Based on our results, baicalin may have applications in the treatment of skin photodamage caused by UVA irradiation.     

Enzymatic activities affecting exogenous nicotinamide adenine dinucleotide in human skin fibroblasts

The fate of nicotinamide adenine dinucleotide (NAD), AMP, and ADP-ribose supplied to intact human skin fibroblasts was monitored, and the concentrations of intra- and extracellular pyridine and purine compounds were determined by HPLC analysis. Two enzymatic activities affecting extracellular NAD were detected on the plasma membrane, one hydrolyzing the pyrophosphoric bond and yielding nicotinamide mononucleotide (nucleotide pyrophosphatase) and the other cleaving the glycoside link and releasing nicotinamide (NAD-glycohydrolase). No AMP or ADP-ribose was found in the extracellular medium of cells incubated with NAD, the former being completely catabolized to hypoxanthine and the latter degraded to adenine and hypoxanthine. © 1996 Wiley-Liss, Inc.     

Regulation of human skin pigmentation and responses to ultraviolet radiation

Pigmentation of human skin is closely involved in protection against environmental stresses, in particular exposure to ultraviolet (UV) radiation. It is well known that darker skin is significantly more resistant to the damaging effects of UV, such as photocarcinogenesis and photoaging, than is lighter skin. Constitutive skin pigmentation depends on the amount of melanin and its distribution in that tissue. Melanin is significantly photoprotective and epidermal cells in darker skin incur less DNA damage than do those in lighter skin. This review summarizes current understanding of the regulation of constitutive human skin pigmentation and responses to UV radiation, with emphasis on physiological factors that influence those processes. Further research is needed to characterize the role of skin pigmentation to reduce photocarcinogenesis and to develop effective strategies to minimize such risks.     

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.     

Comparison of DNA adduct levels associated with exogenous and endogenous exposures in human pancreas in relation to metabolic genotype

Recently, we examined normal human pancreas tissue for DNA adducts derived from either exogenous chemical exposure and/or endogenous agents. In an effort to explain the different types and levels of DNA adducts formed in the context of individual susceptibility to cancer, we have focused on gene-environment interactions. Here, we report on the levels of hydrophobic aromatic amines (AAs), specifically those derived from 4-aminobiphenyl (ABP), and DNA adducts associated with oxidative stress in human pancreas. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been performed. Using the same DNA, the genotypes were determined for N-acetyltransferase 1 (NAT1), the glutathione S-transferase (GST) M1, GSTP1, GSTT1, and NAD(P)H quinone reductase-1 (NQO1) as possible modulators of adduct levels because their gene products are involved in the detoxification of AAs, lipid peroxidation products and in redox cycling. These results indicate that ABP-DNA adducts, malondialdehyde-DNA adducts, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) adducts are present at similar levels. Of the metabolic genotypes examined, the presence of ABP-DNA adducts was strongly associated with the putative slow NAT1*4/*4 genotype, suggesting a role for this pathway in ABP detoxification.     

Kinetics of ultraviolet induced DNA excision repair in rat and human fibroblasts

To obtain more information on the well-documented low excision-repair capacity of rodent cells in comparison with human cells, we have studied this form of DNA repair in UV-irradiated human and rat skin fibroblasts. For this purpose, we have determined (i) unscheduled DNA synthesis (UDS), using autoradiography, (ii) the number and size of repaired sites with the bromodeoxyuridine (BrdU) photolysis assay and (iii) the removal of Micrococcus luteus UV-endonuclease susceptible sites (ESS). We found rat cells to be quite capable of performing DNA-repair synthesis, as demonstrated by both UDS and BrdU photolysis, whereas they almost completely lacked the capacity to remove pyrimidine dimers, as indicated by the persistence of ESS. This discrepancy will be discussed in terms of the types of mechanisms by which mammalian cells may recognize and remove UV-induced photoproducts.     

Anethole prevents hydrogen peroxide-induced apoptosis and collagen metabolism alterations in human skin fibroblasts

Thyroid hormone stimulates erythropoietic differentiation. However, severe anemia is sometimes seen in patients with hyperthyroidism, and the mechanisms have not been fully elucidated. Bone marrow is comprised about 2–8 % oxygen, and the characteristics of hematopoietic stem cells have been shown to be influenced under hypoxia. Hypoxia-inducible factor-1 is a critical mediator of cellular responses to hypoxia and an important mediator in signal transduction of thyroid hormone [triiodothyronine (T3)]. The aim of this study was to investigate the effect of T3 on erythropoiesis under hypoxia mimicking physiological conditions in the bone marrow. We maintained human erythroleukemia K562 cells under hypoxic atmosphere (2 % O₂) and examined their cellular characteristics. Compared to that under normal atmospheric conditions, cells under hypoxia showed a reduction in the proliferation rate and increase in the hemoglobin content or benzidine-positive rate, indicating promotion of erythroid differentiation. T3 had no effect on hypoxia-induced erythroid differentiation, but significantly inhibited activin A/erythroid differentiation factor-induced erythroid differentiation. Moreover, GATA2 mRNA expression was suppressed in association with erythroid differentiation, while T3 significantly diminished that suppression. These results suggest that T3 has a direct suppressive effect on erythroid differentiation under hypoxic conditions.     

Antimutagenesis as organism defense system against damaging factors of endogenous and exogenous origin

Antimutagenic system is represented as the system combining all enzymatic and metabolic processes of the organism at all levels. The problem of supporting of autoantimutagenic system with antimutagenic preparations is discussed. A number of possible mechanisms of antimutagenic action of different substances are considered.     

DNA-protein cross-linking by ultraviolet radiation in normal human and xeroderma pigmentosum fibroblasts.

DNA-protein cross-linking by ultraviolet radiation was measured in human fibroblasts by an adaptation of the method of DNA alkaline elution. To measure cross-linking, a controlled frequency of DNA single-strand breaks was introduced by exposing the cells to a low dose of X-ray at 0 degrees C prior to analysis by alkaline elution. The effect of prior exposure of the cells to ultraviolet radiation was to reduce the rate and/or extent of DNA elution from X-irradiated cells. This effect was attributed to DNA-protein cross-linking, since the effect was reversed by treatment of the cell lysates with proteinase-K. Cross-linking in normal human fibroblasts occurred immediately after ultraviolet irradiation, prior to the appearance of DNA single-strand breaks due to excision repair. Upon incubation of normal cells after exposure, to ultraviolet radiation, the cross-linking was partially repaired. In xeroderma pigmentosum cells, cross-links appeared as in normal cells, but there was no repair. Instead, the extent of cross-linking appeared to increase upon incubation after ultraviolet irradiation.     

人皮肤成纤维细胞在紫外线B照射后的TGF—β和HSP70表达

目的和方法：采用人体皮肤成纤维细胞,观察了紫外线照射后细胞TGF－β表达与HSP70表达水平的相关性。结果：①经不同剂量的紫外线B照射后,TGF－βmRNA表达与HSP70表达水平呈正相关（r=0.906);②应用抗TGF－βⅡ型受体抗体后,在紫外线B照射后细胞培养上清液中的TGF－β含量与细胞HSP70表达水平呈负相关（r=-0.995)。结论：在紫外线B照射诱导人皮肤成纤维细胞表达HSP70的反应过程另TGF－β参与其信号转导。     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.